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Journal
of
Pathology
Journal of Pathology
and T ranslational M edicine
and
Translational Medicine
May 2021
Vol. 55 / No.3
jpatholtm.org
pISSN: 2383-7837
eISSN: 2383-7845
PD-L1 Assessment in
Urothelial Carcinoma
Journal of Pathology and
Translational Medicine
Vol. 55, No.3, pp 163-237, May 2021
Journal of Pathology and Translational Medicine
Volume 55 • Number 3 • May 2021 (bimonthly)
J Pathol Transl Med
Published since 1967 pISSN: 2383-7837
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Front cover image: Differences in scoring algorithm and heterogeneous results of various PD-L1 arrays in urothelial carcinoma (p165, 168).
© Copyright 2021 by the Korean Society of Pathologists/the Korean Society for Cytopathology
Journal of Pathology and Translational Medicine is an Open Access journal under the terms of the Creative Commons Attribution Non-Commercial License
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This paper meets the requirements of KS X ISO 9706, ISO 9706-1994 and ANSI/NISO Z.39.48-1992 (Permanence of Paper).
J Pathol Transl Med
Journal of Pathology and Translational Medicine pISSN: 2383-7837
eISSN: 2383-7845
Volume 55, Number 3, May 2021
CONTENTS
REVIEWS
163 Programmed cell death-ligand 1 assessment in urothelial carcinoma: prospect and limitation
Kyu Sang Lee, Gheeyoung Choe
171 Hepatocellular adenomas: recent updates
Haeryoung Kim, Young Nyun Park
181 olecular biomarker testing for non–small cell lung cancer: consensus statement of the Korean Cardiopulmonary Pathology
M
Study Group
Sunhee Chang, Hyo Sup Shim, Tae Jung Kim, Yoon-La Choi, Wan Seop Kim, Dong Hoon Shin, Lucia Kim, Heae Surng Park, Geon Kook Lee, Chang Hun Lee,
Korean Cardiopulmonary Pathology Study Group
ORIGINAL ARTICLES
192 I dentification of PI3K-AKT signaling as the dominant altered pathway in intestinal type ampullary cancers
through whole-exome sequencing
Niraj Kumari, Rajneesh K. Singh, Shravan K. Mishra, Narendra Krishnani, Samir Mohindra, Raghvendra L.
202 ismatch repair deficiency and clinicopathological characteristics in endometrial carcinoma:
M
a systematic review and meta-analysis
Alaa Salah Jumaah, Hawraa Sahib Al-Haddad, Mais Muhammed Salem, Katherine Ann McAllister, Akeel Abed Yasseen
212 Prognostic role of ALK-1 and h-TERT expression in glioblastoma multiforme: correlation with ALK gene alterations
Dalia Elsers, Doaa F. Temerik, Alia M. Attia, A. Hadia, Marwa T. Hussien
CASE REPORTS
225 S pindle cell oncocytoma of the sella turcica with anaplastic features and rapid progression in short-term follow-up:
a case report with proposal of distinctive radiologic features
Dong Ja Kim, SangHan Lee, Mee-seon Kim, Jeong-Hyun Hwang, Myong Hun Hahm
CONTENTS CONTINUED
© 2021 The Korean Society of Pathologists/The Korean Society for Cytopathology
230 Hepatoid thymic carcinoma: a case report of a rare subtype of thymic carcinoma Ji-Seon Jeong, Hyo Jeong Kang, Uiree Jo, Min Jeong Song, Soon Yeol Nam, Joon Seon Song EDITORIALS 235 Histologic subtyping of ampullary carcinoma for targeted therapy Seung-Mo Hong 236 Prognostic and predictive markers in glioblastoma and ALK overexpression Jang-Hee Kim Instructions for Authors for Journal of Pathology and Translational Medicine are available at http://jpatholtm.org/authors/authors.php © 2021 The Korean Society of Pathologists/The Korean Society for Cytopathology
Journal of Pathology and Translational Medicine 2021; 55: 163-170
https://doi.org/10.4132/jptm.2021.02.22 REVIEW
Programmed cell death-ligand 1 assessment in urothelial carcinoma:
prospect and limitation
Kyu Sang Lee1,2, Gheeyoung Choe1,2
1
Department of Pathology, Seoul National University Bundang Hospital, Seongnam;
2
Department of Pathology, Seoul National University College of Medicine, Seoul, Korea
Programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) inhibition has revolutionized the treatment paradigm of uro-
thelial carcinoma (UC). Several PD-L1 assays are conducted to formulate appropriate treatment decisions for PD-1/PD-L1 target thera-
py in UC. However, each assay has its own specific requirement of antibody clones, staining platforms, scoring algorithms, and cutoffs
for the determination of PD-L1 status. These prove to be challenging constraints to pathology laboratories and pathologists. Thus, the
present article comprehensively demonstrates the scoring algorithm used and differences observed in each assay (22C3, SP142, and
SP263). Interestingly, the SP142 score algorithm considers only immune cells and not tumor cells (TCs). It remains controversial whether
SP142 expressed only in TCs truly accounts for a negative PD-L1 case. Moreover, the scoring algorithm of each assay is complex and
divergent, which can result in inter-observer heterogeneity. In this regard, the development of artificial intelligence for providing assis-
tance to pathologists in obtaining more accurate and objective results has been actively researched. To facilitate efficiency of PD-L1 test-
ing, several previous studies attempted to integrate and harmonize each assay in UC. The performance comparison of the various PD-L1
assays demonstrated in previous studies was encouraging, the exceptional concordance rate reported between 22C3 and SP263. Al-
though these two assays may be used interchangeably, a clinically validated algorithm for each agent must be applied.
Key Words: Urothelial carcinoma; Programmed cell death-ligand 1; 22C3; SP142; SP263; Immunotherapy
Received: January 26, 2021 Accepted: February 22, 2021
Corresponding Author: Gheeyoung Choe, MD, PhD, Department of Pathology, Seoul National University Bundang Hospital, 82 Gumi-ro 173beon-gil, Bundang-gu, Seongnam 13620,
Korea
Tel: +82-31-787-7711, Fax: +82-31-787-4012, E-mail: gychoe@snu.ac.kr
The U.S. Food and Drug Administration (FDA) has approved and SP142)—each of which involves different antibody clones,
the use of programmed cell death protein 1/programmed death- autostainers, scoring algorithms, and cutoffs [2,3]. This com-
ligand 1 (PD-1/PD-L1) inhibitors (pembrolizumab, nivolumab, plexity implicated in the usage of PD-L1 assays has raised ques-
atezolizumab, and durvalumab) in the treatment of various can- tions on their comparability and interchangeability. Although
cers. PD-1/PD-L1 target therapies are no longer limited to tumor previous studies have attempted to integrate and harmonize the
subtypes or origins. The interesting emerging concept of ‘PD- PD-L1 assays in non-small cell lung cancer (NSCLC), discordant
Loma’ refers to tumors that respond to PD-1/PD-L1 target ther- PD-L1 expression was observed across the results of various assays
apy [1]. Urothelial carcinoma (UC) is one of the most significant [4-6]. Similarly, in UC, although a good correlation between
PD-Lomas. Particularly, pembrolizumab and atezolizumab are each assay was observed, none exhibited a perfect agreement
indicated as first-line treatments in patients with locally advanced [3,7-9].
or metastatic UC who are not eligible for cisplatin-containing Diagnostic assays can be essential for the use of therapeutics
chemotherapy and whose tumors are PD-L1 immunohisto- (companion diagnostics) or may inform on improving the bene-
chemistry (IHC)-positive. PD-L1 IHC is a pivotal diagnostic fit without restricting drug access (complementary diagnostics)
technique used for determining the necessity of PD-1/PD-L1 [10]. Notably, 22C3 and SP142 were companion diagnostics in
target therapy. All agents are FDA-approved, used in conjunction the first-line use of pembrolizumab and atezolizumab, respectively
with one of the PD-L1 assays available (22C3, 28-8, SP263, [11,12]. However, 28-8 and SP263 have not been used as com-
pISSN 2383-7837 © 2021 The Korean Society of Pathologists/The Korean Society for Cytopathology
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/
eISSN 2383-7845 by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 163
164 • Lee KS et al.
panion diagnostics for nivolumab and durvalumab in advanced and/or cytoplasmic staining (at any intensity) were considered
UC patients [13,14]. Thus, the interpretation of 22C3 and ‘IC-positive.’ PD-L1 expression and CPS are suggested to be
SP142 may be crucial in practice and should be carefully assessed evaluated at higher magnification (20 ×). Infiltrating UC, high-
by pathologists. PD-L1 expression in immune cells (ICs) is grade papillary UC, carcinoma in situ, and metastatic UC are
comparatively as significant as that in tumor cells (TCs) in UC. included under CPS, whereas low-grade papillary UC and tumor
The correlation between IC PD-L1 expression and treatment necrotic area should be excluded. Finally, 22C3 is defined as
response has been demonstrated in all clinical studies conducted positive if CPS ≥ 10 in UC.
on UC, except in the case of nivolumab/28-8 [14].
In this review, we have discussed the scoring algorithm and Ventana SP142
differences in each PD-L1 assay in detail (Table 1) and assessed SP142 is scored as the proportion of tumor area that is occu-
the current issues posed by PD-L1 testing in UC. Since the 28-8 pied by PD-L1–expressing ICs at any intensity (Table 1, Fig. 1).
assay is rarely used in most countries, including South Korea, Unlike 22C3, SP142 measures the area occupied instead of the
22C3, SP142, and SP263 in UC were evaluated. number of stained cells. ICs include lymphocytes, macrophages,
dendritic cells, and granulocytes, wherein stained ICs can be
COMPARISON OF PD-L1 ASSAY found as aggregates in intratumoral or contiguous peritumoral
INTERPRETATION IN stroma, or as single cell spread among TCs. Tumor area for PD-
UROTHELIAL CARCINOMA L1 scoring is defined as the area occupied by viable TCs and their
associated intratumoral and contiguous peritumoral stroma. In
Agilent 22C3 papillary UC, the stroma in fibrovascular cores is considered intra-
According to the 22C3 (pharmDx) interpretation manual, tumoral stroma. Tumor necrosis should be excluded for scoring.
PD-L1 expression was determined by using the combined posi- SP142 staining at any intensity of tumor-infiltrating ICs covering
tive score (CPS) in UC, which is the number of PD-L1–stained ≥ 5% of the tumor area is considered positive.
cells (TC + IC) divided by the total number of viable TCs, and
multiplied by 100 (Table 1, Fig. 1) [15]. The result of the calcu- Ventana SP263
lation can exceed 100; however, the maximum score is defined as According to the manufacturer’s manual, SP263 status is deter-
CPS 100. The CPS is defined accordingly: mined by the percentage of TCs with any membrane staining, or
by the percentage of tumor-associated ICs with staining at any
CPS = Number
of PD-L1 staining cells (TCs + ICs)/ intensity (Table 1, Fig. 1). Similar to SP142, SP263 expresses the
Total number of viable TCs × 100 area proportionate to the tumor area measured. The percentage
of tumor area occupied by any tumor-associated ICs (Immune
ICs include lymphocytes and macrophages, but do not include Cells Present, ICP) is used to determine IC expression, which is
plasma cells, neutrophils, and eosinophils. TCs with partial or defined as the percentage area of ICP exhibiting PD-L1–positive
complete linear membrane staining (at any intensity) were con- IC staining. SP263 status is considered positive if any of the fol-
sidered ‘TC-positive.’ ICs within the tumor nests and/or the im- lowing criteria are met:
mediately adjacent supporting stroma with convincing membrane - ≥ 25% of the TCs exhibit membrane staining; or,
Table 1. Comparison of PD-L1 assays for UC and difference in scoring algorithm
22C3 SP142 SP263
Manufacturer Agilent Ventana Ventana
Drug Pembrolizumab Atezolizumab Durvalumab
Status Companion diagnostic Companion diagnostic Complementary diagnostic
Scoring algorithm CPS = #TC+ and #IC+/Total #TC × 100 ≥ 10 IC+/tumor area ≥ 5% TC+/tumor area or ≥ 25%
ICP > 1%: IC+/ICP ≥ 25% or
ICP = 1%: IC+/ICP = 100%
Algorithm based on Positive cell number Positive cell area Positive cell area
Cell type Tumor cells, lymphocytes, and macrophages Lymphocytes, macrophages, dendritic Tumor cells, lymphocytes, macrophages,
cells and granulocytes histiocytes, plasma cells, and neutrophils
PD-L1, programmed cell death-ligand 1; UC, urothelial carcinoma; CPS, combined positive score; TC, tumor cell; IC, immune cell; ICP, immune cells present.
https://jpatholtm.org/ https://doi.org/10.4132/jptm.2021.02.22
PD-L1 assessment in urothelial carcinoma • 165
Yellow area (IC+)
SP142 = × 100 ≥ 5(%)
Red area
A
TC+ count in navy
+
IC+ count in yellow
22C3(CPS) = × 100 ≥ 10
TC count in red area
B
Navy area (TC+)
SP263 = × 100 (%) ≥ 25 (%)
Red area
or
Yellow area (IC+)
= × 100 (%) ≥ 25 (%)
Orange are (ICP)
C
Fig. 1. Representative pictures for the comparison of programmed death-ligand 1 (PD-L1) assays and differences in scoring algorithm of
urothelial carcinoma (UC). (A) Scoring algorithm of SP142 is based on the proportion of tumor area that is occupied by PD-L1-expressed im-
mune cells (ICs) at any intensity. (B) Scoring algorithm of 22C3 is determined by using the combined positive score (CPS) in UC, which is the
number of PD-L1–stained cells (tumor cell [TC] plus IC) divided by the total number of viable TCs, multiplied by 100. (C) SP263 status is de-
termined by the percentage of TCs obtained by performing any membrane staining or by the percentage of tumor-associated ICs obtained
by staining at any intensity. SP263-expressed TC area proportion of the tumor area is determined. Also, the percentage of tumor area occu-
pied by any tumor-associated IC (Immune Cells Present, ICP) is used to determine IC expression, and IC positivity is defined as the percent-
age of PD-L1–positive IC area in ICP.
- ICP > 1% and IC+ ≥ 25%; or, or complete circumferential pattern. TC cytoplasmic staining is
- ICP = 1% and IC+ = 100%. disregarded when determining PD-L1 expression. The percentage
The manufacturer’s manual suggests that an expression level of tumor-associated IC with staining is evaluated in addition to TC
greater than or equal to 25% of the TCs or ICs should be consid- staining. Interestingly, IC scoring includes lymphocytes, mac-
ered significant. Membrane staining of TCs can exhibit a partial rophages, histiocytes, plasma cells, and neutrophils. IC staining is
https://doi.org/10.4132/jptm.2021.02.22 https://jpatholtm.org/
166 • Lee KS et al.
assessed by initially reviewing the entire tumor area and by deter- carefully so as to ensure that no patient is devoid of treatment
mining the ICP. Subsequently, the percentage of PD-L1 expressing opportunity.
ICs within the ICP is visually estimated (IC+). Additionally, in
cases where the percentage of ICP in the tumor area is 1%, it is INTER-OBSERVER HETEROGENEITY
considered positive only when 100% of the ICs are stained.
Inter-observer variability may lead to the obtainment of dis-
INTERCHANGEABILITY OF PD-L1 ASSAYS cordant results for PD-L1, which can consequentially impact
IN UROTHELIAL CARCINOMA therapy decisions. A recent study demonstrated that inter-ob-
server agreement for each assay is moderate to high for IC stain-
The use of different expensive autostainers and various assays ing (0.532–0.729) as well as TC staining (0.609–0.883) based on
is neither economical nor reasonable for pathology laboratories. intra-class correlation coefficient obtained for UC [20]. However,
Interchangeability of different assays may enable the usage of 22C3 and 28-8 exhibited low inter-observer correlation in IC
only one standardized PD-L1 assay in laboratories. In NSCLC, staining, while SP142 showed low inter-observer correlation in TC
Adam et al. [16] showed that 28-8, 22C3, and SP263 assays dem- staining [20]. Downes et al. [21] suggested that excellent inter-
onstrated close analytical performance for TC staining across seven observer agreement could be found using SP263 and 22C3,
centers. However, a significant discrepancy was observed between whereas PD-L1 scoring using SP142 was associated with a
SP142 and the other three assays for TC staining, whereas IC higher level of subjectivity in head and neck squamous cell car-
staining results were similar [17-19]. Moreover, the SP142 assay cinoma, breast carcinoma, and UC. The study of inter-observer
was an outlier that detected markedly less PD-L1 expression in heterogeneity of PD-L1 assays has also been well conducted using
both TCs and ICs [5]. NSCLC samples. According to Cooper et al. [22], 10 patholo-
Rijnders et al. [3] have suggested that agreement in the PD- gists reported good reproducibility at both 1% cutoffs of 22C3,
L1 status in UC between 22C3, 28-8, SP142, and SP263 is whereas agreement was slightly lower for the 50% cutoff. More-
substantial (80%–90%), implying that these assays may be inter- over, the Cardiopulmonary Pathology Study Group of the Korean
changeable in clinical practice. Moreover, a collaborative study Society of Pathologists investigated the inter-observer heteroge-
conducted by the Russian Society of Clinical Oncology and the neity of PD-L1 staining with 22C3 using NSCLC samples [23].
Russian Society of Pathology found that a patient with UC clas- Inter-observer reproducibility for the 1% cutoff was found to be
sified as negative by one of the three tests (22C3, SP142, and relatively lower than the 50% cutoff, in contrast to the results
SP263) using the corresponding cutoff rule was highly likely reported by Cooper et al. [22]. Similarly, Rimm et al. [5] indi-
(91%–100%) to be classified as negative based on the results of cated that 13 pathologists reported excellent concordance when
any other test performed, therefore avoiding the need for repeated scoring TCs stained with any antibody (22C3, 28-8, SP142, and
testing [8]. Furthermore, Zajac et al. [9] reported a high level of E1L3N) but reported poor concordance when scoring ICs stained
analytical concordance among the SP263, 22C3, and 28-8 assays with any antibody using NSCLC samples [16]. Although dif-
for TC and IC staining; however, such a level of concordance fering results were reported in previous studies of both UC and
was not observed for SP142. Additionally, Hodgson et al. [7] NSCLC, inter-observer heterogeneity of PD-L1 seems to occur.
demonstrated that SP142 TC staining intensity was lower in UC
and hypopharyngeal squamous cell carcinoma samples, although CORRELATION BETWEEN PD-L1 EXPRESSION
there existed adequate analytic comparability between 22C3 and AND HISTOLOGIC SUBTYPES
SP263. Another recent study has highlighted greater differences
in the assays used for the analysis of PD-L1–stained TCs, particu- UC is among the most histologically diverse cancers. A pre-
larly between SP142 and other assays [20]. These analytical find- vious study found that infiltrating UC exhibits significantly
ings were consistent with other studies conducted using NSCLC higher T cell infiltration and PD-L1 expression than non-invasive
samples, which suggested that SP142 did not exhibit sufficient papillary UC and UC in situ [24]. In addition to the conventional
concordance with the other three assays. While 22C3 and SP263 morphology observed, UC can contain elements of squamous
have a high concordance rate and can be used interchangeably, differentiation, glandular differentiation, nested, plasmacytoid,
clinical validation for each immune-therapy remains a necessity sarcomatoid, and/or rarer variants. UC patients with histologic
[9]. The interchangeability of PD-L1 assay must be considered variants account for up to one-third of advanced cases. Li et al.
https://jpatholtm.org/ https://doi.org/10.4132/jptm.2021.02.22
PD-L1 assessment in urothelial carcinoma • 167
[24] found that PD-L1 was expressed in a significant percentage culties in analyses performed using PD-L1 assays. First, it is dif-
of histologic variant of UC cases (cutoff 1% TC, 37% to 54%; ficult to distinguish between TC and IC positivity. Representa-
cutoff 5% TC, 23% to 37%), while the highest PD-L1 expres- tively, distinguishing between TCs and macrophages proves
sion was observed in patients with UC exhibiting squamous dif- burdensome when PD-L1 is stained, because macrophages are
ferentiation [25]. These results suggest that patients with histo- of comparative size to the TCs (Fig. 2H). Reviewing the hema-
logic variants of UC may benefit more from anti–PD-1/PD-L1 toxylin and eosin-stained slides can be helpful in this respect.
therapy. 22C3 should be used to count both ICs and TCs, which may
not be problematic; however, SP142 should be excluded PD-
DISCUSSION L1–positive TCs. Moreover, ICs commonly include lympho-
cytes and macrophages. However, the ICs of SP263 additionally
PD-1/PD-L1 target therapy has garnered considerable atten- include plasma cells and neutrophils. Although this can also
tion as a potential treatment strategy for patients with advanced prove to be beneficial if we compare the hematoxylin and eosin-
UC. These agents are approved by the U.S. Food and Drug Ad- stained slides with PD-L1–stained slides, it remains uncertain
ministration for the treatment of patients with locally advanced whether PD-L1–positive neutrophils, plasma cells, and lym-
or metastatic UC, with disease progression during or following phocytes can be distinguished effectively (Fig. 2I). Lastly, the
platinum-containing chemotherapy, or disease progression within lamina propria at the base of the papillary lesion may contain
12 months of neoadjuvant or adjuvant treatment with platinum- lymphoid aggregates that show PD‑L1 positivity, whereas only
containing chemotherapy. Moreover, pembrolizumab and atezoli- the lamina propria contiguous to the base of the tumor is consid-
zumab have received approval for first-line treatment of locally ered part of the tumor area. Moreover, in fragmented tissue sam-
advanced or metastatic UC in patients ineligible for cisplatin- ples, including transurethral resection or biopsies—where distinc-
containing chemotherapy. PD-L1 expression levels in UC can tion of intra- or peritumoral stroma cannot be ascertained—only
thus effectively aid physicians in identifying patients who are stroma that is contiguous to individual tumor nests is included
more likely to benefit from anti–PD-1/PD-L1 therapy. in the tumor area definition (Fig. 2G). However, the meaning
However, every single agent is tested in conjunction with a spe- of ‘contiguous’ is ambiguous and subjective.
cific PD-L1 assay, which must be performed on a specific staining The 22C3 scoring system uses the CPS algorithm. Theoreti-
platform. Moreover, the PD-L1 scoring algorithm is heteroge- cally, regardless of whether the number of TCs is large or small,
neous and unique for each assay. These aspects have encouraged 22C3 should be used to count all TCs present in the tumor area
pathologists to consider assay interchangeability. Several studies for the denominator. Additionally, all PD-L1–positive TCs and
have attempted to harmonize PD-L1 assays conducted for NSCLC ICs should be counted in the tumor area for obtaining values
and UC samples. However, SP142 was an outlier that detected for the numerator. This scoring algorithm is labor-intensive and
markedly less PD-L1 expression in TC (Fig. 2A–C) [5-7,9,18,20]. an accurate calculation is practically impossible for the whole
In contrast, relatively high concordance was observed between tumor area. Thus, the 22C3 manufacturer (Agilent) suggests
SP263 and 22C3. As each assay is performed using different that a partial portion of the tumor can be selected and scored.
immunogens, and thus a unique epitope, different PD-L1 confor- However, these results may be inconsistent due to intratumoral
mations or isoforms may lead to the obtainment of heterogeneous heterogeneity of 22C3 expression.
results. Moreover, the location of the antibody-binding domain has A comparatively higher positive cutoff (≥ 25% of TC) is required
been known to affect the staining pattern, resulting in increased for SP263. If the test results do not meet the TC cutoff, the IC
variability [26]. This discordance in the results suggests that cutoff (≥ 25% of IC) can be evaluated subsequently. Unlike SP142,
the prospects for interchangeability of the assays is not optimistic. the total tumor area is not evaluated for IC scoring. IC positivity
Although the discordance rate between SP263 and 22C3 is low, is only evaluated in the ICP. Therefore, SP263 results may easily
there is no scientific evidence to prove that two assays can be used meet the cutoff (≥ 25% of IC) because ICP is relatively smaller
interchangeably. It is of utmost priority that all patients should than the total tumor area values used as a denominator. However,
receive proper treatment, without any exceptions arising due to the evaluation of complex geographic ICP may pose challenges.
assay discordance. For accurate evaluation, one must physically draw and cut out
The scoring algorithm for each PD-L1 assay in UC is described the ICP—which lies beyond the confines of plausibility. Hence,
in Table 1 and Fig. 1. There are several common practical diffi- advances in artificial intelligence and digital pathology are nec-
https://doi.org/10.4132/jptm.2021.02.22 https://jpatholtm.org/
168 • Lee KS et al.
22C3 SP142 SP263
A B C
SP142 22C3
D E F
22C3 SP142 SP142
G H I
Fig. 2. (A–C) Representative pictures of heterogeneity observed in programmed death-ligand 1 (PD-L1) assay results (22C3, SP142, and
SP263). SP142 was an outlier that detected markedly less PD-L1 expression in tumor cells (TCs). (D–F) Biopsy sample of metastatic urothe-
lial carcinoma with PD-L1 expression observed only in TCs. Although SP142 was expressed in TCs, the result obtained was negative. How-
ever, the result for 22C3 was positive. This patient may be administered with pembrolizumab, but not with atezolizumab, as first-line therapy.
(G) It is difficult to distinguish between the various subtypes of immune cells. If the regions indicated by the red arrows are plasma cells, they
should be excluded from the 22C3 score. (H) Investigation may be necessary to ascertain whether the SP142-stained cells are TCs or ICs.
This picture shows SP142 expression on intra-tumoral macrophages. (I) In fragmented tissue samples, including transurethral resection or
biopsy samples, where the distinction between intratumoral or peritumoral stroma cannot be clearly observed. The yellow area contiguous
to the base of the tumor is considered part of the tumor area.
essary for the precise assessment of SP263 expression. At present, herein were SP142-negative, these patients demonstrated a com-
SP263 is not clinically used as a companion diagnostic for dur- plete response to atezolizumab. Whilst these results were obtained
valumab in UC. for only a limited number of cases, it should nonetheless be con-
SP142 evaluation for the determination of the IC-positive sidered whether the exclusion of positive TCs from the scoring
area and score seems relatively simpler than performing other algorithm is a reasonable methodology. The ability of SP142 to
assays. However, exclusion of SP142 TC positivity information detect TC expression is low, which may lead to the generation
from scoring may not be an appropriate approach. We analyzed of statistical bias in clinical trials of atezolizumab in UC. This is
three UC biopsy cases (unpublished data), which showed only probably main reason that TC were not included in the scoring
strong PD-L1 positivity in TCs and not in ICs, with negative algorithm of the SP142. Re-evaluation of SP142 TC expression
SP142 results (Fig. 2D–F). Although all the UC cases studied may thus be necessary in a novel clinical study to assess anti–PD-1/
https://jpatholtm.org/ https://doi.org/10.4132/jptm.2021.02.22PD-L1 assessment in urothelial carcinoma • 169
PD-L1 therapy in UC. Furthermore, in cases where SP142 ex- proach to overcome these challenges. Moreover, in the future,
pression is observed only in TCs, it is recommended to adopt digital pathology and artificial intelligence may assist PD-L1
the 22C3 test to determine the applicability of pembrolizumab evaluation with greater accuracy.
as first-line treatment.
These complex score algorithms and intratumoral heteroge- Ethics Statement
Not applicable.
neity of PD-L1 expression can result in inter-observer heteroge-
neity, particularly in scoring the SP142 of UC [20,21]. It is not Availability of Data and Material
difficult to score definite PD-L1 positive or negative cases; how- Data sharing not applicable to this article as no datasets were generated or
ever, inter-observer heterogeneity must be observed in cases with analyzed during the study.
approximate cutoff scores (e.g., 22C3 CPS, 5%–15%; SP142,
Code Availability
3%–10%, and so on). Providing training to the pathologist in Not applicable.
these aspects as well as in the use of artificial intelligence may
be a possible method for reduction of the inter-observer PD-L1 ORCID
discordance. Kyu Sang Lee https://orcid.org/0000-0003-2801-9072
Gheeyoung Choe https://orcid.org/0000-0001-6547-5603
Several studies have suggested the prognostic significance of
PD-L1 expression in various malignancies; PD-L1–expressing Author Contributions
tumors tend to exhibit poor prognosis [27]. Previous meta- Conceptualization: KSL, GC. Data curation: KSL. Formal analysis: KSL.
Investigation: KSL. Methodology: KSL. Supervision: GC. Writing—original
analyses have demonstrated that PD-L1 expression is correlated draft: KSL. Writing—review & editing: KSL, GC. Approval of final manu-
with worse prognosis and advanced clinicopathological features script: all authors.
in UC [28,29]. Moreover, Kawahara et al. indicated that UC with
Conflicts of Interest
high-grade features exhibited higher PD-L1 expression [30]. A
The authors declare that they have no potential conflicts of interest.
recent study has reported that the increased expression of PD-
L1 is correlated with histologic variants of UC, including squa- Funding Statement
mous, glandular, plasmacytoid, and sarcomatoid differentiation No funding to declare.
[25]. Histologic variants of UC constitute a high-grade feature
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https://doi.org/10.4132/jptm.2021.02.27 REVIEW
Hepatocellular adenomas: recent updates
Haeryoung Kim1, Young Nyun Park2
1
Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul;
2
Department of Pathology, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, Seoul, Korea
Hepatocellular adenoma (HCA) is a heterogeneous entity, from both the histomorphological and molecular aspects, and the resultant
subclassification has brought a strong translational impact for both pathologists and clinicians. In this review, we provide an overview of
the recent updates on HCA from the pathologists’ perspective and discuss several practical issues and pitfalls that may be useful for di-
agnostic practice.
Key Words: Hepatocellular adenoma; Classification; Pathology
Received: February 4, 2021 Revised: February 27, 2021 Accepted: February 28, 2021
Corresponding Author: Young Nyun Park, MD, PhD, Department of Pathology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea
Tel: +82-2-2228-1678, Fax: +82-2-362-0860, E-mail: young0608@yuhs.ac
Hepatocellular adenoma (HCA) is defined as a benign mono- dence of HCA in men, and the incidence of oral contraceptive use
clonal proliferation of hepatocytes [1]. It is more prevalent in was lower in female patients [2,3,5]. The main clinical implica-
Western countries, with an incidence of 3–4 cases/100,000 in tions of HCA include the risk of bleeding, especially in larger
Europe and North America [1], and its incidence is much lower tumors, and the risk of malignant transformation to hepatocel-
in Asian countries [2-6]. Since the first characterization of the lular carcinoma (HCC). The risk of HCC development depends
molecular subtypes of HCAs in 2006 [7], HCA has now become on the subtype, being the highest in β-catenin-activated HCAs
an increasingly heterogeneous entity, and this has brought a (B-HCAs). The general indications for surgical management of
strong translational impact for pathologists (Table 1) [8-10]. In HCAs include male gender, large size (>5 cm), interval growth
this review, we will summarize the clinicopathological and molec- during follow up on imaging, and atypical pathological features
ular characteristics of the various subtypes of HCA, and discuss the (e.g., atypical cytoarchitectural features, presence of β-catenin
various pitfalls in the pathological diagnosis of HCA. activation).
In general, HCAs are grossly well demarcated but non-encap-
GENERAL CLINICOPATHOLOGICAL FEATURES sulated, and the color varies from pale yellow-tan to bile-stained
OF HEPATOCELLULAR ADENOMA depending on the histology. Hemorrhage or peliosis may be pres-
ent. HCAs are more frequently solitary lesions; however, multiple
The typical patient is female and of reproductive age (15–50 HCAs may occur, and the term “adenomatosis” is used when
years), while HCAs are relatively rare in men, children and elderly there are 10 or more HCAs. In cases of multiple HCAs, most cases
patients (> 65 years). The major risk factors for HCA include oral demonstrate multiple HCAs of the same molecular subtype,
contraceptive use, obesity, metabolic syndrome, alcohol intake although some tumors from the same patient may belong to dif-
and use of anabolic steroids. Other conditions associated with ferent subgroups of HCA [11]. The size is variable, ranging from
HCA development include glycogen storage diseases (especially 1cm to as large as 30 cm. Unlike HCCs, the background liver is
types 1 and 3), galactosemia, tyrosinemia, familial polyposis coli, typically non-cirrhotic, although advanced stage fibrosis may be
polycystic ovary syndrome and β-thalassemia. Interestingly, a present in the setting of alcoholic liver disease, metabolic syndrome
few series from Asian countries have demonstrated a higher inci- or vascular disorders. Histologically, the tumor cells resemble he-
pISSN 2383-7837 © 2021 The Korean Society of Pathologists/The Korean Society for Cytopathology
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/
eISSN 2383-7845 by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 171172 • Kim H et al.
Table 1. Summary of the clinicopathological and molecular characteristics of different HCA subtypes
Characteristic features
Subtype (frequency, %)
Molecular Clinical Histopathological Immunohistochemical
HNF1A-inactivated HCA HNF1A inactivating mutations Female, obesity, MODY3, Diffuse steatosis LFABP expression loss
(30%–40%) (germline 10%, somatic 90%) adenomatosis
Inflammatory HCA gp130/IL6ST, FRK, STAT3, GNAS, Obesity, metabolic syndrome, Sinusoidal dilatation SAA, CRP expression
(40%–50%) JAK1 mutations alcohol, oral contraceptives Vascular proliferation
Inflammatory cell
infiltration
Ductular reaction
Focal steatosis
β-catenin–activated
HCA (10%)
β-catenin CTNNB1 exon 3 activating Male, young age, anabolic Cytological and Nuclear β-catenin expression
(exon 3)–activated mutations steroids, glycogen storage architectural atypia Diffuse strong GS expression
HCA (7%) disease, increased risk of
HCC transformation
β-catenin CTNNB1 exon 7 or 8 activating Low risk of HCC - Absent/rare nuclear β-catenin
(exon 7,8)–activated mutations transformation expression
HCA (3%) GS expression: absent/weak/
patchy
β-catenin–activated gp130/IL6ST, STAT3, FRK, GNAS, Similar to inflammatory HCA Similar to inflammatory SAA, CRP expression
inflammatory HCA JAK1 mutations + CTNNB1 exon Increased risk of HCC HCA Nuclear β-catenin, diffuse
(5%–10%) 3 or 7/8 mutations transformation (ex.3) Cytoarchitectural atypia strong GS expression (ex.3)
(ex.3)
Sonic hedgehog– INHBE-GLI1 fusion, resulting Obesity, hemorrhage Hemorrhage PTGDS, ASS1
activated HCA (4%) in sonic hedgehog pathway
activation
Unclassified HCA (< 7%) Unknown - - -
HCA, hepatocellular adenoma; MODY3, maturity-onset diabetes type 3; LFABP, liver fatty acid binding protein; SAA, serum amyloid A; CRP, C-reactive pro-
tein; HCC, hepatocellular carcinoma; GS, glutamine synthetase; PTGDS, prostaglandin D2 synthase; ASS1, argininosuccinate synthase 1.
patocytes, demonstrating eosinophilic or clear cytoplasm, and they abolic syndrome, alcoholic liver disease etc.).
are arranged in thin trabeculae. Portal tracts are absent, and thin- Immunohistochemistry for serum amyloid A (SAA) and C-
walled vascular channels and small arteriolar structures are seen. reactive protein (CRP) may provide important diagnostic clues,
as IHCA is characterized by the overexpression of these acute
RECENT UPDATES ON THE CLASSIFICATION phase reactants via STAT3 activation. SAA and/or CRP expres-
OF HEPATOCELLULAR ADENOMA sion in IHCA is usually diffuse and strong, and sharply demar-
cated from the surrounding liver parenchyme. However, it should
Inflammatory HCA be noted that the adjacent liver may be focally or even diffusely
Inflammatory HCA (IHCA) is the most common subtype positive for SAA and/or CRP in some cases, especially when there
(40%–50%) of HCAs, and demonstrates constitutive activation is marked inflammation or hemorrhage in the background liver,
of the interleukin-6/JAK/STAT pathway. Molecular alterations and in the setting of previous embolization [12]. Therefore, the
include mutations in gp130/IL6ST (50%), FRK (10%), STAT3 histological context should be taken into account when inter-
(5%), GNAS (5%), ROS1 (3%), and JAK1 (1%). The main risk preting SAA/CRP stains and it is important that the staining
factors for IHCA include obesity, metabolic syndrome and alcohol results are compared with the background liver, preferably also
intake. with positive control tissues.
The main histological features include foci of inflammation, Importantly, about 10% of IHCA also demonstrate muta-
thick arteries, and sinusoidal dilatation (Fig. 1). Congestion, tions in CTNNB1 (B-IHCA, “mixed” HCA). Although the
hemorrhage and peliosis may be present. Portal tracts are absent; risk for HCC transformation is generally low in IHCA, the con-
however, ductular reaction and pseudoportal tracts are frequently currence of strong β-catenin activation in IHCAs increases the
seen. Steatosis may be observed and is usually focal. The back- risk of HCC development. Therefore, the addition of β-catenin
ground liver frequently demonstrates steatosis, which may be and glutamine synthetase (GS) immunohistochemistry is also
attributed to the patient’s underlying risk factors (obesity, met- necessary, in order to identify mixed HCAs (B-IHCA). B-HCAs
https://jpatholtm.org/ https://doi.org/10.4132/jptm.2021.02.27Hepatocellular adenoma updates • 173
A B C
D E F
Fig. 1. Inflammatory hepatocellular adenoma. Sinusoidal dilatation, congestion and peliosis is seen in the tumor (A–C). Inflammatory cell infil-
tration (C), thick arteriolar structures with ductular reaction, resembling portal tracts (“pseudo-portal tracts”) and steatosis (D) may be seen in
these tumors. Diffuse steatosis may be seen in the background liver (A). The tumor cells express serum amyloid A (E; inset: granular cyto-
plasmic staining in tumor cells) and C-reactive peptide (F).
are described in more detail in the following section. of Bex7,8-HCA are still unclear, although the risk of HCC devel-
opment appear to be low in these tumors, unlike the Bex3-HCAs
β-catenin–activated HCA [10,13,14]. CTNNB1 alterations may also occur in a subset of
Approximately 10% of HCAs demonstrate CTNNB1 muta- IHCAs (B-IHCA or “mixed” HCA, 5%–10%).
tions/deletions leading to different levels of β-catenin pathway The characteristic clinical features associated with Bex3-HCAs
activation. These tumors are designated as B-HCA. CTNNB1 al- include male gender, a history of androgen administration and
terations most often occur in exon 3 (7%, Bex3-HCA) or in exons underlying glycogen storage disease. These tumors are usually
7 and 8 (3%, Bex7,8-HCA). Mutations or large deletions in exon solitary and rarely multiple. Mild cytological atypia may be ob-
3 most frequently involve the β-Trcp consensus site (D32–S37, served in Bex3-HCAs, and architectural atypia, including mild
also known as the exon 3 hotspot) and these are associated with trabecular thickening, small cell change or pseudoglandular
high levels of β-catenin activation and high risk of HCC trans- structures, is also frequently seen (Fig. 2). Bile or lipofuscin
formation [13,14]. Outside of the β-Trcp consensus site, T41 pigments are frequently observed in the tumor cells; the lipofus-
and S45 mutations in exon 3 have also been frequently demon- cin pigmentation may be very prominent in some HCAs [15].
strated, and these are associated with moderate to weak levels of Most importantly, as Bex3-HCAs are associated with high risk of
β-catenin activation. In contrast, mutations in CTNNB1 exon malignant transformation to HCC, the practical implication of
7 (K335) and exon 8 (W383, R386, and N387) result in weak this subtype of HCA is in excluding the possibility of well-differ-
β-catenin activation. The clinical and histopathological features entiated HCC. This differential diagnosis is discussed later.
https://doi.org/10.4132/jptm.2021.02.27 https://jpatholtm.org/174 • Kim H et al.
A B C
D E
Fig. 2. β-catenin–activated hepatocellular adenoma (B-HCA). There is mild trabecular thickening (A) and pseudoglandular structures (B, C).
Mild cytological atypia is present (A–C). Cholestasis may be seen (C). On immunohistochemistry, B-HCA with strong β-catenin activation
(Bex3-HCA) demonstrates nuclear β-catenin expression (D), instead of the normal membranous pattern (inset), and diffuse homogeneous
glutamine synthetase expression is seen in such tumors (E, inset: higher-power view).
Although direct sequencing of the CTNNB1 gene would be onstrate diffuse heterogeneous GS expression (50%–90% of tu-
the most definitive means of characterizing the molecular sub- mor cells expressing GS in a starry-sky pattern). Bex7,8-HCAs lack
type of a presumed B-HCA, this is not feasible in most clinical the immunohistochemical features of strong β-catenin activation
practices. Fortunately, immunohistochemical stains for β-catenin (i.e., diffuse strong GS expression, nuclear β-catenin expression).
and GS have been demonstrated to be good surrogate markers Weak patchy GS staining in addition to the “normal” perivenular
reflecting CTNNB1 status. Nuclear β-catenin expression and dif- pattern has been frequently seen in association with weak β-catenin
fuse strong homogeneous GS expression are the typical immuno- activation (absent or rare nuclear β-catenin), which is common in
histochemical features of B-HCAs with strong β-catenin activa- Bex7,8-HCAs, but also rarely seen in Bex3-HCAs with S45 muta-
tion (non-S45 Bex3-HCA). Interestingly, GS expression patterns tions. Interestingly, GS accentuation and discontinuous band-like
have been recently demonstrated to reflect the mutational status GS staining at the tumor border has been described in Bex3-HCAs
of CTNNB1 (Fig. 3). Diffuse homogeneous expression (strong with S45 mutations and Bex7,8-HCAs, respectively [16,17]. These
GS expression in > 90% of tumor cells) has been demonstrated expression patterns are different from the “map-like” GS pattern
in Bex3-HCAs with mutations or large deletions in the D32–S37 of focal nodular hyperplasia (FNH), which is described later. In
hotspot of CTNNB1 exon 3 (β-Trcp consensus site). In contrast, contrast, GS expression in the normal liver has a perivenular dis-
Bex3-HCAs with CTNNB1 exon 3 T41 or S45 mutations, which tribution (“normal” pattern), where GS expression is limited to 1
have been associated with moderate β-catenin activation, dem- to 3 layers of hepatocytes around the central vein.
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